1. Field of the Invention
The invention relates to a confocal laser scanning microscope with an illuminating configuration, which provides an illuminating beam for illuminating a specimen region, with a scanning configuration, which guides the illuminating beam over the specimen while scanning, and with a detector configuration, which via the scanning configuration images the illuminated specimen region by means of a confocal aperture on to at least one detector unit.
2. Related Art
Confocal laser scanning microscopes of the type initially mentioned are known in the state of the art, for example, let us cite the German patent DE 197 02 753 A1 in reference thereto. Most recently, components and technical systems from microscopy, specifically from confocal imaging laser scanning microscopes, have been ever more frequently applied to spectroscopic imaging techniques. In this manner, it is possible to survey the spectroscopic characteristics of a selected specimen region without destroying or touching the specimen region. Confocal optic microscopy thereby makes it possible to selectively detect optical signals, which are generated within a diffractionally limited confocal volume whose magnitude lies in the realm of micrometers. Laser scanning microscopes with scanning laser beams and/or with specimen feed units can generate high focal resolution for two or three dimensional representations of the specimen under examination. Owing to this characteristic, confocal laser scanning microscopy has nearly asserted itself as the standard for fluorescent specimens in the field of biomedical technology.
Based on the highly exponential power of the chemical contents, confocal Raman microscopy is very attractive, particularly with regards to its application. The patent EP 0 542 962 B1 describes a setup for confocal Raman microscopy in which the appropriate selection of a spacially resolving two-dimensional detector is used to produce the condition of confocality.
A problem in Raman spectroscopy consists in that the levels of signal intensity are frequently lower by several orders of magnitude as compared to classical fluorescence spectroscopy. In practice, the integration time per measuring point frequently exceeds 1 minute. The periods of measurement resulting from that often span many hours or days, which naturally sets narrow limits on the capabilities of confocal Raman spectroscopy for recording two or three dimensional microscopic images with high dot density. A reduction in the integration time would theoretically be conceivable by increasing laser output performance, however, this quickly leads to the destruction of the specimen.
One is faced with similar problems in the area on non-linear optical microscopy, which, for example, offers attractive contrasting methods with the second harmonic generation but whose practical application however, is highly limited as well due to the low intensity of the signals and to the long measurement periods associated therewith.
A confocal microscope setup is known from the U.S. Pat. No. 6,134,002 in which a spectral analyzer is used as the detector. The analyzer admits the radiation through an entrance slit, whereby the linear slit region corresponds to a line region on the specimen. A dotted image is scanned on the specimen. The radiation to be detected is decoupled to the analyzer via a beam splitter acting as the primary color splitter, said beam splitter being located either between two scan mirrors of a scanning unit or being arranged in front of the scanning unit as seen in the direction toward the specimen. In the first-named variant, the dotted image on the specimen scanned by the two scan mirrors is only descanned in one spatial direction so that the spectral analyzer is impinged by radiation scanned along one line. In the second variant, the radiation is completely descanned by the scan mirrors and thereby comes to a rest state and can therefore be once more expanded with a cylindrical optical system toward a pinhole. The construction known from the U.S. Pat. No. 6,134,002 achieves an acceleration in image recording speed by shortening the periods of spectral analysis, which is why it forcibly depends on a spectral analyzer with a slit-shaped entrance region serving as a detector, thus it is highly limited with regards to a plurality of possible detectors. The initially mentioned problems associated with high laser performance are also a given factor in the design according to the U.S. Pat. No. 6,134,002.